Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/principal finding

Using "middle aged" mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1(+)CD45(-) cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1(+)CD45(-) cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1(+)CD45(-) cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/significance

These studies demonstrate that cloned Sca-1(+)CD45(-) cells derived from CSs from infarcted "middle aged" hearts are enriched for second heart field (i.e., Isl-1(+)) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.

Background

Aberrant Ca²⁺ handling is a prominent feature of heart failure. Elucidation of the molecular mechanisms responsible for aberrant Ca²⁺ handling is essential for the development of strategies to blunt pathological changes in calcium dynamics. The peptidyl-prolyl cis-trans isomerase peptidyl-prolyl isomerase 1 (Pin1) is a critical mediator of myocardial hypertrophy development and cardiac progenitor cell cycle. However, the influence of Pin1 on calcium cycling regulation has not been explored. On the basis of these findings, the aim of this study is to define Pin1 as a novel modulator of Ca²⁺ handling, with implications for improving myocardial contractility and potential for ameliorating development of heart failure.

Methods and results

Pin1 gene deletion or pharmacological inhibition delays cytosolic Ca²⁺ decay in isolated cardiomyocytes. Paradoxically, reduced Pin1 activity correlates with increased sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and Na²⁺/Ca²⁺ exchanger 1 protein levels. However, SERCA2a ATPase activity and calcium reuptake were reduced in sarcoplasmic reticulum membranes isolated from Pin1-deficient hearts, suggesting that Pin1 influences SERCA2a function. SERCA2a and Na²⁺/Ca²⁺ exchanger 1 associated with Pin1, as revealed by proximity ligation assay in myocardial tissue sections, indicating that regulation of Ca²⁺ handling within cardiomyocytes is likely influenced through Pin1 interaction with SERCA2a and Na²⁺/Ca²⁺ exchanger 1 proteins.

Conclusions

Pin1 serves as a modulator of SERCA2a and Na²⁺/Ca²⁺ exchanger 1 Ca²⁺ handling proteins, with loss of function resulting in impaired cardiomyocyte relaxation, setting the stage for subsequent investigations to assess Pin1 dysregulation and modulation in the progression of heart failure.

Vascular networks within a living organism are complex, multi-dimensional, and challenging to image capture. Radio-angiographic studies in live animals require a high level of infrastructure and technical investment in order to administer costly perfusion mediums whose signals metabolize and degrade relatively rapidly, diminishing within a few hours or days. Additionally, live animal specimens must not be subject to long duration scans, which can cause high levels of radiation exposure to the specimen, limiting the quality of images that can be captured. Lastly, despite technological advances in live-animal specimen imaging, it is quite difficult to minimize or prevent movement of a live animal, which can cause motion artifacts in the final data output. It is demonstrated here that through the use of postmortem perfusion protocols of radiopaque silicone polymer mediums and ex-vivo organ harvest, it is possible to acquire a high level of vascular signal in preclinical specimens through the use of micro-computed tomographic (microCT) imaging. Additionally, utilizing high-order rendering algorithms, it is possible to further derive vessel morphometrics for qualitative and quantitative analysis.

Background

MicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive.

Methods and results

We hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p<0.05) and contractile protein expression without affecting their electrophysiological properties (p>0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased I(to), I(Ks) and I(Kr), and decreased I(f) (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca(2+) transient amplitude and kinetics. Molecular pathway analyses were performed for further insights.

Conclusion

We conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.